The long-term objective of this proposal is to uncover critical clues towards innovative and effective interventions for acute renal failure secondary to ischemia-reperfusion injury (IRI), which continues to represent a common and devastating problem in clinical medicine. The proposed studies are based on the finding that apoptosis represents a major mechanism leading to tubule cell death following IRI, and downregulation of specific apoptotic pathways may offer a unique approach to the amelioration of IRI in both the native and the transplanted kidney. Genome-wide screening techniques and follow-up proteomic analyses by our group have shown that Daxx, a novel and controversial apoptosis-related factor, is significantly induced in tubule epithelial cells following early IRI. Recent studies in other cell types have identified an intriguing Daxx/ASK1/JNK signal transduction pathway that may be pro- or anti-apoptotic. This pathway in the kidney remains unexplored. Our hypothesis is that Daxx activates unique cell death pathways in the kidney following IRI, which may be targeted for innovative therapies. The specific aims of this focused proposal will test this hypothesis by identifying: Specific Aim 1: The direct role of Daxx in mediating tubule cell damage following IRI, Specific Aim 2: The role of ASK1, an effector molecule downstream of Daxx, in regulating tubule cell damage following IRI, and Specific Aim 3: The role of JNK, an effector molecule downstream of ASK1, in regulating tubule cell damage following IRI. We will utilize a spectrum of complementary techniques in cell culture and animal models to analyze the structural, biochemical and functional responses to IRI following manipulation of the Daxx/ASK1/JNK pathways. These include inhibitory maneuvers such as small interfering RNAs, gain-of-function methods such as forced over-expression, and loss-of-function models such as JNK knockout mice. The ability of pharmacologic agents such as inhibitors of ASK1 and JNK to alter the morphologic and functional responses to IRI will also be determined in cell culture and animal models. Collectively, the results of this focused proposal will provide important insights into the molecular pathogenesis of apoptotic and necrotic cell death in IRI. The long term goal is to identify novel ASK1- and JNK-based pharmacologic interventions aimed at ameliorating renal cell damage and preventing renal failure secondary to ischemia-reperfusion injury.